Ubiquitin is a protein modifier that plays a central role in cellular regulation and disease processes. It is covalently attached to proteins through an enzymatic cascade involving three classes of enzymes (E1, E2, E3 enzymes), with the E3s conferring target protein specificity. Among the several classes of E3 enzymes, HECT E3s are unique in that they function via a covalent enzyme-ubiquitin intermediate and participate directly in catalysis of ubiquitin conjugation. First discovered through studies on HPV-associated cervical cancer, HECT E3s also play important roles in other types of cancer, neurologic disease, various signaling pathways, and intracellular protein trafficking. One human HECT E3, Herc5, catalyzes conjugation of an interferon-induced ubiquitin-like modifier called ISG15, which is important in anti-viral responses. These diverse health-related functions highlight the importance of understanding the mechanism, function, and regulation of HECT E3s. Aim 1 of this renewal application will focus on critical aspects of the biochemical mechanism of HECT E3s. A complete understanding of enzyme mechanism is critical for development of small molecules (drugs) that can either up- or down-regulate HECT E3 function in specific disease states. Importantly, very few natural targets of HECT E3s have been identified, and this has hampered our understanding of their biological functions. In Aim 2, we will develop and implement a completely novel approach for HECT E3 substrate identification, based on the unique mechanism and structure of the catalytic domain. Aim 3 represents a new research direction, where we have applied approaches developed in our ISG15 studies to analyze Co-Translational Ubiquitination (CTU), a phenomenon that has been linked to protein quality control, protein folding, and protein aggregation diseases. We will address the function of CTU through identification of target proteins and characterization of the enzymes of the pathway. We anticipate that these studies will have a strong and sustained impact on the many aspects of human health that are affected by the ubiquitin system, including cancer biology, cell signaling pathways, protein folding diseases, and infectious diseases.